Abstract

In this study, we compared the pharmacological and biochemical characteristics of A_2B adenosine receptors in recombinant (hA_2BHEK293 cells) and native cells (neutrophils, lymphocytes) by using a new potent 8-pyrazole xanthine derivative, [³H]N-benzo[1,3]dioxol-5-yl-2-[5-(1,3-dipropyl-2,6-dioxo-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yl-oxy]-acetamide] ([³H]MRE 2029-F20), that has high affinity and selectivity for hA_2B versus hA₁,hA_2A, and hA₃ subtypes. [³H]MRE 2029-F20 bound specifically to the hA_2B receptor stably transfected in human embryonic kidney (HEK) 293 cells with K_D of 2.8 ± 0.2 nM and B_max of 450 ± 42 fmol/mg of protein. Saturation experiments of [³H]MRE 2029-F20 binding in human neutrophils and lymphocytes detected a single high-affinity binding site with K_D values of 2.4 ± 0.5 and 2.7 ± 0.7 nM, respectively, and B_max values of 79 ± 10 and 54 ± 8 fmol/mg of protein, respectively, in agreement with real-time reverse transcription polymerase chain reaction studies showing the presence of A_2B mRNA. The rank order of potency of typical adenosine ligands with recombinant hA_2B receptors was consistent with that typically found for interactions with the A_2B subtype and was also similar in peripheral blood cells. 5′-N-Ethyl-carboxamidoadenosine stimulated cAMP accumulation in both hA_2BHEK293 and native cells, whereas phospholipase C activation was observed in recombinant receptors and endogenous subtypes expressed in neutrophils but not in lymphocytes. MRE 2029-F20 was revealed to be a potent antagonist in counteracting the agonist effect in both signal transduction pathways. In conclusion, [³H]MRE 2029-F20 is a selective and high-affinity radioligand for the hA_2B adenosine subtype and may be used to quantify A_2B endogenous receptors. In this work, we demonstrated their presence and functional coupling in neutrophils and lymphocytes that play a role in inflammatory processes in which A_2B receptors may be involved.